本文選題：Y染色體 + STR��； 參考：《四川大學(xué)》2003年博士論文

【摘要】： 目的 為了提高Y染色體遺傳標(biāo)記的個(gè)人識(shí)別能力和非父排除能力，尋找新的Y-STR和建立新一代遺傳標(biāo)記Y-SNP的檢測(cè)方法十分必要。本課題旨在闡明8個(gè)新的Y-STR的等位基因結(jié)構(gòu)和單倍型頻率，為法醫(yī)學(xué)應(yīng)用提供基礎(chǔ)。建立用普通高效液相色譜儀檢測(cè)單鏈寡聚核苷酸的方法，為檢測(cè)單堿基引物延伸反應(yīng)的產(chǎn)物提供定量分析基礎(chǔ)。建立單堿基引物延伸反應(yīng)，掌握單堿基引物延伸反應(yīng)的一般規(guī)律，為法醫(yī)學(xué)Y-SNP分析提供基礎(chǔ)。方法 在數(shù)據(jù)庫(kù)中尋找到8個(gè)新的Y-STR基因座；利用軟件重新設(shè)計(jì)其中的兩個(gè)基因座，即DYS443和DYS444的引物。應(yīng)用PCR方法擴(kuò)增Y-STR，電泳分型；對(duì)所有基因座的等位基因進(jìn)行測(cè)序，構(gòu)建等位基因分型標(biāo)準(zhǔn)物，按國(guó)際法醫(yī)遺傳學(xué)會(huì)(ISFG)原則命名等位基因。在普通的高效液相色譜儀上，，建立檢測(cè)合成的單鏈寡聚核苷酸方法。利用耐熱DNA聚合酶催化單堿基引物延伸反應(yīng)，建立2個(gè)Y-SNP的單堿基引物延伸反應(yīng)。結(jié)果 等位基因測(cè)序結(jié)果提示DYS443、DYS453、DYS455、DYS456是簡(jiǎn)單重復(fù)序列Y-STR，而DYS444、DYS448、DYS457、DYS458是復(fù)雜重復(fù)序列Y-STR；在我們研究的群體樣本中，DYS443、 DYS444、DYS448、DYS453、DYS455、DYS456、DYS457、DYS458的 基因變異度分別為0.7742、0.7671、0.7453、0.3545、0.0549、0.6988、 0.5058、0.8213。單倍型變異度為0.9991，其個(gè)人識(shí)別能力和非父排除率 也為0.9991。在普通的高效液相色譜儀上，應(yīng)用反相離子對(duì)高效液相色 譜法成功地分離了4種長(zhǎng)度不同的單鏈寡聚核昔酸(18、19、20、Zlnt) 以及兩種序列不同的19nt單鏈寡聚核昔酸。用己知濃度的單鏈寡聚核營(yíng) 酸作為標(biāo)準(zhǔn)品，制作了濃度與峰面積的標(biāo)準(zhǔn)曲線。建立了分析2個(gè)丫SNP 的單堿基引物延伸反應(yīng)，掌握了單堿基引物延伸反應(yīng)的規(guī)律。應(yīng)用建立 的HPLC方法檢測(cè)單堿基引物延伸反應(yīng)的產(chǎn)物，并且對(duì)產(chǎn)物進(jìn)行了定量。 結(jié)論DYS443和DYS444的引物經(jīng)重新設(shè)計(jì)后，擴(kuò)增片段長(zhǎng)度縮短，有 利于準(zhǔn)確分型，結(jié)果證明引物設(shè)計(jì)是成功的。分析了這些基因座的等位 基因序列，為按照ISFG的推薦原則進(jìn)行標(biāo)準(zhǔn)化命名提供了依據(jù)。單倍型 變異度表明這8個(gè)丫STR具有較好的個(gè)人識(shí)別能力和非父排除率，能夠 較好地提高Y染色體遺傳標(biāo)記的個(gè)人識(shí)別和非父排除能力。在普通高效 液相色譜儀上，利用離子對(duì)反相高效液相色譜方法能夠分辨長(zhǎng)度、序列 不同的單鏈寡聚核昔酸，其分離度與寡聚核昔酸的序列和長(zhǎng)度都有關(guān)， 為檢測(cè)單堿基引物延伸反應(yīng)的產(chǎn)物提供了基礎(chǔ)。通過(guò)建立2個(gè)Y-SNP的 SNuPE反應(yīng)，了解SNuPE反應(yīng)的一般規(guī)律，揭示單堿基引物延伸反應(yīng)的 關(guān)鍵是選擇合適的DNA聚合酶及合適的引物和模板的比例。為丫SNP 應(yīng)用于法醫(yī)學(xué)提供了方法學(xué)基礎(chǔ)。
[Abstract]:Objective in order to improve the individual recognition ability and non paternal exclusion ability of Y chromosome genetic markers, it is necessary to find new Y-STR and establish a new generation of genetic marker Y-SNP. The purpose of this study is to clarify 8 new Y-STR allelic structures and haplotype frequencies, and to provide a basis for forensic application. The method of detecting single strand oligonucleotides by Chromatograph provides a basis for quantitative analysis of the product of single base primer extension reaction. The general rule of single base primer extension reaction is established, and the general rule of single base primer extension reaction is grasps, which provides the basis for Y-SNP analysis of forensic medicine. Methods 8 NEW Y-STR loci were found in the data base; The two primers, DYS443 and DYS444, were redesigned by software. The PCR method was used to amplify the Y-STR and electrophoresis typing; the alleles of all the loci were sequenced and the allele standard was constructed, and the alleles were named according to the international forensic genetic association (ISFG) principle. On the ordinary high performance liquid chromatograph, it was established. The single strand oligonucleotide method was detected. 2 Y-SNP single base primer extension reactions were established by using the heat resistant DNA polymerase to catalyze the single base primer extension reaction. The results of allele sequencing showed that DYS443, DYS453, DYS455, DYS456 were simple repeat sequences Y-STR, while DYS444, DYS448, DYS457, DYS458 were complex repeat sequences. In the group samples we studied, DYS443,
DYS444, DYS448, DYS453, DYS455, DYS456, DYS457, DYS458
Gene variability was 0.7742,0.7671,0.7453,0.3545,0.0549,0.6988.
0.5058,0.8213. haplotype variability was 0.9991, its personal recognition ability and non parent exclusion rate.
Also for 0.9991., the reversed-phase ion pair high performance liquid chromatography is used on the ordinary high performance liquid chromatograph.
4 different lengths of single chain oligonucleotide (18,19,20, Zlnt) were successfully separated by spectral analysis.
And two kinds of 19nt single chain oligonucleotides with different sequences.
Acid as a standard product, the standard curve of concentration and peak area was made. 2 SNP were analyzed.
The single base primer extension reaction has mastered the rule of single base primer extension reaction.
The HPLC method was used to detect the products of single base primer extension reaction, and the products were quantified.
Conclusion after DYS443 and DYS444 primers were redesigned, the length of the amplified fragments was shortened.
The results showed that primer design was successful. The alleles of these loci were analyzed.
The gene sequence provides a basis for standardized naming according to the recommendation principle of ISFG. Haplotype
The variation degree indicates that these 8 STR have better personal recognition ability and non father exclusion rate.
It can improve the ability of personal identification and non parent elimination of Y chromosome genetic markers.
On the liquid chromatograph, the ion pair RP HPLC method can distinguish length and sequence.
The separation degree of different single chain oligonucleic acid is related to the sequence and length of oligonucleotide.
It provides a basis for detecting the products of single base primer extension reaction. Through the establishment of 2 Y-SNP
SNuPE reaction, understand the general rule of SNuPE reaction, and reveal the single base primer extension reaction.
The key is to select suitable DNA polymerase and suitable primers and template ratio. For ya SNP
It provides a methodological basis for the application of forensic medicine.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2003
【分類號(hào)】：D919

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 侯一平,張思仲;溫度調(diào)控高效液相色譜探索人類基因組變異的進(jìn)展[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2000年03期

2 廖林川,孟海英,侯一平,張思仲,顏有儀,蘇智廣,李英碧,吳瑾,張霽;用溫度調(diào)控高效液相色譜探索基因組單核苷酸多態(tài)性的方法研究[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2000年03期

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